A century of scientific research has established that mitochondria dysfunction is one of the most prevalent and profound phenotypes of human cancer cells. At the genetic level, the differences between normal and tumor cells include both the depletion of mitochondrial DNA (mtDNA) and somatic mtDNA mutations in all human cancers examined to date. The rate of mutation in mtDNA appears to be 20 times higher than that in nuclear DNA. This high rate of mutation is due to the high concentration of reactive oxygen species (ROS) produced by the mitochondria, limited DNA repair and lack of DNA protective histones in the mitochondria. Until now, studies have focused on the identification and characterization of mutations in mtDNA, with little insight into the contribution of these mutations to the etiology of cancer. To understand the contribution of mtDNA in etiology of cancer, we have generated the complete mtDNA- knockout ( rho(o)) in breast epithelial cells. We demonstrate that mtDNA knockout in epithelial cells leads to neoplastic transformation that shows increased invasiveness and anchorage-independent growth. Interestingly, these properties of (rho(o) epithelial cells are restored by reintroduction of mtDNA (transmitochondrial cybrid) suggesting a role for mtDNA in the induction of the cancer phenotype. We also demonstrate that mitochondrial gene knockout leads to i) gross chromosomal rearrangements (GCR) 2) resistance to apoptosis 3) altered expression of ROS generating NADPH oxidase (Nox1) involved in tumorigenesis. Based on these observations we hypothesize that mutant mtDNA contributes to the etiology of cancer. MtDNA knockout cells established in our laboratory will serve as a novel tool for transfer of mutant mtDNA in a uniform genetic background. The mtDNA mutations could contribute to neoplastic transformation of epithelial cells by increasing oxidative stress, modulating cell proliferation, modulating apoptosis, affecting the expression of ROS producing Noxi protein and increasing nuclear genome instability. To test this hypothesis we plan to: i) Construct a mtDNA mutator system and characterize those mutants that are found in primary breast tumors. 2) Generate isogenic mutant mtDNA cybrids, and determine the consequences of mutations on mitochondrial function. 3) Using isogenic transmitochondrial cybrids determine the consequences of mtDNA mutations on cell proliferation, apoptosis and nuclear genome instability. 4) Using isogenic transmitochondrial cybrids determine the importance of superoxide generating Nox1 in tumorigenesis. 5) Using isogenic mutant mtDNA cybrids assess the significance of mtDNA mutations on in vitro and in vivo tumorigenesis and metastasis in severe combined immunodeficient (SCID) mice. The proposed study will help determine the contribution of mutant mtDNA to the development of human cancer.